DNA methylation landscapes from pig’s limbic structures underline regulatory mechanisms relevant for brain plasticity

Epigenetic dynamics are essential for reconciling stress-induced responses in neuro-endocrine routes between the limbic brain and adrenal gland. CpG methylation associates with the initiation and end of regulatory mechanisms underlying responses critical for survival, and learning. Using Reduced Representation Bisulfite Sequencing, we identified methylation changes of functional relevance for mediating tissue-specific responses in the hippocampus, amygdala, hypothalamus, and adrenal gland in pigs. We identified 4186 differentially methylated CpGs across all tissues, remarkably, enriched for promoters of transcription factors (TFs) of the homeo domain and zinc finger classes. We also detected 5190 differentially methylated regions (DMRs, 748 Mb), with about half unique to a single pairwise. Two structures, the hypothalamus and the hippocampus, displayed 860 unique brain-DMRs, with many linked to regulation of chromatin, nervous development, neurogenesis, and cell-to-cell communication. TF binding motifs for TFAP2A and TFAP2C are enriched amount DMRs on promoters of other TFs, suggesting their role as master regulators, especially for pathways essential in long-term brain plasticity, memory, and stress responses. Our results reveal sets of TF that, together with CpG methylation, may serve as regulatory switches to modulate limbic brain plasticity and brain-specific molecular genetics in pigs.

Differences in global methylation rates across tissue comparisons.The boxplot panels show the differences in the distribution of the methylation rates. The difference in their distances was quantified using a twosample Kolmogorov-Smirnov test. D, the maximum vertical distance between the cumulative distributions. All pairwise differences were significantly different (p-value < 2.2e-16). The scattered plot shows the principal component analysis using mean methylation rates from DMRs, suggesting tissue as the major influencing factor.
Supplementary Figure 1: a. Differences in the distribution of the methylation rates across hypothalamus, hippocampus, amygdala, and adrenal gland. b. Principal component analysis using two factors: tissue and sex.
This protein-protein association STRING network 1 shows the inter-connectivity between 226 genes that we identified harbor at least one meCpGs their promoter region (TSS ± 2kb) and that exhibit significant changes in the methylation (q-value < 0.01) rate among the hypothalamus, hippocampus, amygdala and adrenal gland from 78 German Landrace piglets. This network reveals a set of genes whose molecular function was enriched for ontology terms associated with direct and indirect DNA gene regulatory functions. To obtain these gene set, we filtered and annotated meCpGs only based on the significance level, independently of the differences in their methylation rate. This network was build using STRING, it shows the full network, but disconnected nodes were hidden. Nodes represent genes, and their color reveals gene association to its regulatory functions. The color of the edges denotes the type of evidence that supports the interaction between pairs of proteins. This network reveals a set of highly interconnected molecular gene regulatory proteins.
Supplementary Figure 2: Protein-protein association STRING network for genes with significantly different meCpGs (q-value < 0.01).
The structure, nodes interconnectivity, and set up of this STRING network 1 are identical to the supplementary figure S4. However, this network uses a different color code to reveal a set of genes with biological function enriched for terms in the context of organism-, neuro-, and cell development and communication, among others. This network only shows parent GO terms for child terms significantly enriched in this gene set (q-value < 0.01). The whole set of enriched child terms is in the Supplementary Table S7. This network also reveals that a community of genes, mostly TF and other co-regulatory proteins present high protein-protein association connectivity and multiple biological roles (upper part).
Supplementary Figure 3: Enriched ontology terms for biological processes of genes with at least one meCpGs with significant changes in their methylation rate (q-value < 0.01).
Supplementary Figure 4: Length of DMRs in nucleotides and number of meCpGs (left panels) and density of meCpGs across DMRs per each pairwise comparison (right panels).
Differentially methylated regions that are unique to pairwise comparisons between the hypothalamus (Hypo), hippocampus (Hippo), amygdala (Amy), and adrenal gland (Agland) tissues from German Landrace pigs. Green and blue indicate an increase or decrease in methylation compared across tissues, respectively (>= 0.1, light, >=0.25, dark). This panel shows differences across brain tissues (top row) and among brain tissues and adrenal gland (bottom row).
Functional annotation of genes harboring at least one DMR in their promoter across pairwise comparisons (only shows enriched terms with a q-value < 0.01). Molecular Function (a) and Biological process terms (b). The size of the bubble represents the significance level, the greater the bubble the smaller the q-value. The color code represents parent terms, and child terms are in the y axis. Missing pairwise comparisons in a and b indicate that enrichment was not detected.
Supplementary Figure 6: Gene ontology enrichment terms for genes harboring a DMRs in their promoter.
DMRs on the promoter region of LIM Homeobox 2 (LHX2). a. Mean methylation rates profiles for hypothalamus, hippocampus, amygdala, and adrenal gland in pig (top). Annotation to genome features (bottom): Differentially methylated regions track (DMRs, red), CpG Islands track (CGI, dark blue), gene annotation to Sscrofa11.1 (light blue), CpG content 100 bp windows (bottom, black).Image built using Integrative genomics viewer (IGV) 2 . b. LHX2 mRNA expression profile in transcripts per million (TPM) across 54 human tissues. c. LHX2 Normalized mRNA expression profile (NX) across 14 brain tissues from pig. Supplementary Figure 7: DMR on the promoter of LHX2 gene and its mRNA tissue expression.
DMRs on the promoter region of Cadherin 22 (CDH22) and cell cycle exit and neuronal differentiation CEND1 protein-coding genes. Mean methylation rates profiles for hypothalamus, hippocampus, amygdala and adrenal gland for CDH22 (a, top) and CEND1 (b, top) in pig. Annotation to genome features (a and b, bottom), differentially methylated regions track (DMRs, red), CpG Islands track (CGI, dark blue), gene annotation to Sscrofa11.1 (light blue), CpG content 100 bp windows (bottom, black). Image built using IGV 2 . c. CEND1 mRNA expression profile in transcripts per million (TPM) across 54 human tissues. d. CEND1 and CDH22 normalized mRNA expression profile (NX) across 14 brain tissues from pigs.
Supplementary Figure 8: DMRs on the promoter of CDH22 and CEND1 genes and mRNA tissue expression DMRs on the promoter region of SKI Family Transcriptional Corepressor 2 (SKOR2) protein-coding gene and mRNA tissue expression. a. Mean methylation rates profiles for hypothalamus, hippocampus, amygdala, and adrenal gland in pig (top). Annotation to genome features (bottom): Differentially methylated regions track (DMRs, red), CpG Islands track (CGI, dark blue), gene annotation to Sscrofa11.1 (light blue), CpG content 100 bp windows. Image built using IGV 2 . b. SKOR2 mRNA expression profile in transcripts per million (TPM) across 54 human tissues. c. SKOR2 normalized mRNA expression profile (NX) across 14 brain tissues from pig.
Supplementary Figure 9: DMRs on the promoter of SKOR2 gene and tissue expression DMRs on the promoter regions of Inhibitory Synaptic Factor 1 INSYN1 and the copper ion binding and oxidoreductase activity LOXL1 protein-coding genes. a. Mean methylation rates profiles for hypothalamus, hippocampus, amygdala, and adrenal gland in pig (top). Annotation to genome features (bottom): Differentially methylated regions track (DMRs, red), CpG Islands track (CGI, dark blue), gene annotation to Sscrofa11.1 (light blue), CpG content 100 bp windows(bottom, black). Image built using IGV 2 . b and c. INSYN1 and LOXL1 mRNA expression profiles in transcripts per million (TPM) across 54 human tissues, respectively. d and e. INSYN1 and LOXL1 normalized mRNA expression profile (NX) across 14 brain tissues from pigs.
Supplementary Figure 10: DMRs on the promoter of INSYN1 and LOXL1 genes.